REPRODUCCION Y MANEJO

The objective here was to identify the type of interaction in the immunoreaction between VLPs and anti-VLP antibodies of IgA or IgG type using dynamic light scattering. Towards this goal, samples of increasing amounts o f antibody and fixed amount of VLPs were analysed.

Expérimentation: VLP (0.84 mg/ml) and IgA antibody (0.6 mg/ml) solutions were thawed to room temperature before use. 20 pi aliquots of the VLP solution were diluted with 130 pi of deionised water filtered through a 0.2 pm filter. 0-56 pi of antibody were added into the diluted VLP solutions. Each antibody-VLP solution was pipetted into a 200 pi volume flow cell (Hellma England Ltd.) and was placed in the cell holder of the DLS instrument. The temperature was 20.1°C and the data collection time was 100 s. Scattering of the laser light by the sample was measured at a 90° angle and converted by the computer software to size distributions based on intensity, weight and number of particles present in the sample. For each sample, five measurements were taken, the size distributions were converted to a histogram so that they could be averaged. This procedure is described in section 2.4.

For the IgG type anti-VLP antibody, the quantities of VLPs and antibody added were chosen to give the same VLP to antibody ratio as for the experiment with the IgA. 200 pi of VLP solution (0.1 mg/ml) were pipetted in a cylindrical flow cell and antibody solution (1.3 mg/ml) was added in 5.5 pi additions. Rather than measuring a

new sample for each level of antibody added, here a light scattering measurement was performed after each addition of antibody to the same sample of VLP. The light scattering experiment parameters were the same as for the IgA antibody and data were processed similarly.

Results: The result of the light scattering measurement was expressed as size distribution based on weight of particles present in each size class, so that the mass change brought to VLPs by the interaction with the antibodies would be best illustrated. Therefore, figures 3.2 and 3.3 show the weight-based size distributions for four different particle to antibody ratios for the IgA and the IgG antibody respectively.

These two figures gave evidence of a different type of interaction beween VLPs and each type of antibody. The IgA antibody led to a second peak appearing that corresponded to particles of a larger size forming. As the antibody dosing was increased, this peak became a larger part of the population, while mean diameter for both peaks increased. These observations suggested that the addition o f the IgA antibody led to two forms of interaction, where some antibody molecules coated the VLP surface while others caused particles to cross link and form larger immune complexes.

The addition of IgG antibody led to a definite increase in VLP size that reached a saturation point, after which the diameter of the immunocomplexes remained constant (figure 3.3). There was no indication of two population o f particles being formed and so it was proposed that this type of antibody only coated the particle surface.

3.2.2 Sizing Antibody of IgA and IgG Type

In an attempt to select the type of antibody most suitable to the purposes o f the light scattering immunoassay, the light scattering properties of the IgA and IgG anti- VLP antibodies were established. Pure IgA and IgG type antibody solutions were sized by DLS, taking care to have approximately the same concentrations of antibody in the two assayed solutions so that the light scattering results could be comparable.

Expérimentation: For the IgA type antibody 100 pi of sample (0.5 mg/ml) were diluted with 100 pi phosphate buffer (O.IM KH2PO4, pH 7.0, filtered through 0.2 pm

Chapter 3. Development o f a DLS-Based Assay_________________________________ 79 The final concentration was 0.25 mg/ml. One measurement of 316 s duration was performed.

For the IgG antibody 60 pi of sample (1.74 mg/ml) were diluted with 470 pi of phosphate buffer. The final concentration was 0.2 mg/ml. One measurement of 900 s duration was followed at an angle of 90° and temperature of 20± 1.5°C. Because the IgG antibody scattered light much less (0.9 Kcps compared to 50.6 Kcps for about the same concentration of IgA) the data colection time was increased so that a reliable size measurement could be returned.

Results: Figures 3.4 and 3.5 show the size distributions for the IgA and IgG type antibodies respectively.

The sizing measurement for the IgA type antibody showed two populations of molecules, having a mean diameter o f 62.4 nm and 239 nm respectively. The IgG antibody showed a typical monodisperse distribution indicating a mean diameter of

14.7 nm. For similar concentrations of antibody sample, the average intensity of light scattered was 50.6 KCps (kilocounts per sec) for the IgA and 0.9 KCps for the IgG.

Based on these results, the IgG type anti-VLP antibody was chosen for the light scattering immunoassay, since only its interaction with the particles would be detected and the unassociated antibody molecules would remain outside the analysis range and would not contribute to the light scattering signal.

3.2.3 Calibration of Assay for Quantification of Virus-Like Particles in Clarified